X-ray crystallography will be used to determine high-resolution structures for cruzain, cruzain mutants, and their complexes with a variety of inhibitors and their derivatives. Our goal is to develop a therapeutic agent for Chagas' disease by inhibition of the major cysteine protease of Trypanosoma cruzi. Structure analysis will be used in conjunction with enzyme kinetics (Dr. Craik) and computational methods (Dr. Cohen) to find, test (Dr. Craik, Dr. McKerrow, Dr. Engel) and synthesize (Dr. Roush) tight-binding and highly-specific inhibitors for cruzain. To this end, we have recently solved the 2.35Angstroms X-ray structure of cruzain by molecular replacement methods. The structure will be refined to convergence and used as a model for computational methods including DOCK3.0 to begin the search for possible inhibitors. A higher resolution structure (2.0Angstroms or better) will be obtained and will provide a more accurate and informative model for the inhibitor search. Methods will be developed for crystallization of a wide variety of complexes between the cruzains and putative inhibitors. A panoply of structures will be determined, involving cruzain bound to different types of inhibitors and their derivatives, and cruzain mutants with inhibitors and substrates. This will provide a detailed map of the substrate binding site. Our in-depth analysis, in which binding at each subsite is carefully evaluated by site-directed mutagenesis, kinetic analyses, theoretical calculations including molecular dynamics simulations and correlations with atomic temperature factors, should yield an effective therapeutic for Chagas' disease as well as elucidating nuances of cysteine protease function.